G protein-mediated signal transduction pathways are involved in the responses of organisms and their constituent cells to a wide variety of stimuli including light, gustants, odorants, hormones, and neurotransmitters. The nature of the response can be equally diverse varying from changes in gene transcription to altered ion channel kinetics. G protein-mediated signal transduction occurs when an agonist binds selectively to its heptahelical receptor (7TM) leading to the activation of a heterotrimeric G protein. These G proteins are composed of alpha, beta and gamma subunits, and when activated they are able to regulate the activity of specific effectors such as adenylyl cyclase (AC) or G protein-coupled inwardly rectifying K+ (Kir3) channels. 7TMs, G proteins and effectors are all membrane-associated proteins, and for decades two opposing hypotheses have vied for acceptance. The predominant hypothesis has been that these proteins move about independently of one another in membranes, and that signal transduction occurs when they encounter each other as the result of random collisions. The contending hypothesis is that signaling is propagated by an organized complex of these proteins. We have employed two fluorescence-based detection and imaging techniques with the goal of determining which of these hypotheses most accurately describes the process of G protein-mediated signal transduction in a living cell. These techniques known as bioluminescent resonance energy transfer (BRET), and bimolecular fluorescence complementation (BiFC) can be used to determine if proteins are associated in a complex and they can provide both spacial and temporal information about the formation and dissolution of these complexes. Both BRET and BiFC involves the exogenous expression of fusion proteins tagged with either luciferase (Luc) or a fluorescent protein (eg. GFP or YFP). BRET occurs when the bioluminescent energy of the Luc tag is transferred to the fluorescent tag causing it to fluoresce. This only occurs if the tags are juxtaposed (less than 100 angstroms apart) because the fusion proteins associate to form a complex. BiFC is based on the observations that peptide fragments consisting of amino acids 1-158 or 159-238 of YFP (YFP(1-158) and YFP(159-238), respectively) are not fluorescent when co-expressed, but if the two fragments can be brought together by fusing them to proteins that associate to form a complex YFP can be reconstituted with restoration of its fluorescent properties. G protein subunits (Gbeta1 and Ggamma2) were tagged with GFP or with the complementary fragments of YFP (GFP-Ggamma2, YFP(1-158)-Gbeta1 and YFP(159-238)-Ggamma2), and beta2-adrenergic receptors (b2AR), AC and Kir3.1 were tagged with Luc (b2AR-Luc, AC-Luc and Kir3.1-Luc). The tagged signaling molecules retained their biological activity. BRET occurred when GFP-Ggamma2 was co-expressed with either Luc-tagged effectors or with b2AR-Luc indicating that these proteins form complexes with each other. AC and Kir3.1 have also been shown to form stable complexes with the b2AR. The beta-adrenergic agonist, isoproterenol, induced a rapid (t1/2 less than 300 msec) increase in BRET between GFP-Ggamma2 and both AC-Luc and the b2AR-Luc with no apparent change in the affinity of GFP-Ggamma2 for its Luc-tagged partner. This suggests that conformational changes induced by receptor activation, rather than recruitment of G protein, is responsible for effector modulation. The agonist-induced increase in BRET was followed by a relatively slow decline in BRET that coincided with a refractory state caused by receptor desensitization. The proclivity of Gbeta to heterodimerize with Ggamma results in reconstitution of YFP fluorescence in cells co-expressed both YFP(1-158)-Gbeta1 and YFP(159-238-Ggamma2). Direct evidence for the simultaneous presence of three individual proteins in the same complex was demonstrated when BRET was observed in cells co-expressing a reconstituted YFP-tagged Gbeta-gamma heterodimer and AC-Luc, and, consistent with the forgoing results, the BRET was increased by treatment of the cells with isoproterenol. In the absence of co-expressed Kir3.4 subunits, Kir3.1 is not targeted to the cell surface. Although there was a robust BRET between Kir3.1-Luc and GFP-Ggamma2 it was not affected by the membrane impermeable agonist isoproterenol. However, an agonist-induced increase in BRET did occur when the membrane permeable beta-adrenergic agonist cimaterol was used. If Kir3.4 was co-expressed with Kir3.1-Luc and GFP-Ggamma2 the Kir3 channels were targeted to the cell surface, and BRET could be increased by isoproterenol. Taken together these results suggest that the b2AR, G proteins and effectors are assembled into a functional complexes before being transported to the plasma membrane. Furthermore, these complexes persist regardless of whether or not the signal transduction pathway is activated by an agonist, and in so doing contribute significantly to the specificity and efficacy of G protein-mediated signal transduction.